ICES Journal of
Marine Science
ICES Journal of Marine Science (2016), 73(3), 582– 595. doi:10.1093/icesjms/fsu231
Contribution to Special Issue: ‘Towards a Broader Perspective on Ocean Acidification Research’
Original Article
Jonathan C. P. Reum 1*‡, Simone R. Alin 2, Chris J. Harvey1, Nina Bednaršek2, Wiley Evans 2,3,
Richard A. Feely 2, Burke Hales 4, Noelle Lucey5, Jeremy T. Mathis 2,3, Paul McElhany 1, Jan Newton6,
and Christopher L. Sabine 2
1
Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric
Administration, 2725 Montlake Blvd. E, Seattle, WA 98112, USA
2
Pacific Marine Environmental Laboratory, National Oceanic and Atmospheric Administration, 7600 Sand Point Way NE, Seattle, WA 98115, USA
3
Ocean Acidification Research Center, School of Fisheries and Ocean Sciences, University of Alaska Fairbanks, 245 O’Neill Bldg, Fairbanks, AK 99775, USA
4
College of Earth, Ocean and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, USA
5
Marine Environment and Sustainable Development Unit ENEA, PO Box 224, La Spezia, Italy
6
Applied Physics Laboratory, University of Washington, Box 355640, Seattle, WA 98105, USA
*Corresponding author: tel: +1 206 860 3204; fax: +1 206 860 3217; e-mail: jonathan.reum@noaa.gov
Present address: Washington Sea Grant, University of Washington, Box 355640, Seattle, WA, USA
‡
Reum, J. C. P., Alin, S. R., Harvey, C. J., Bednaršek, N., Evans, W., Feely, R. A., Hales, B., Lucey, N., Mathis, J. T., McElhany, P.,
Newton, J., and Sabine, C. L. Interpretation and design of ocean acidification experiments in upwelling systems in the context
of carbonate chemistry co-variation with temperature and oxygen. – ICES Journal of Marine Science, 73: 582 –595.
Received 22 July 2014; revised 24 November 2014; accepted 26 November 2014; advance access publication 7 January 2015.
Coastal upwelling regimes are some of the most productive ecosystems in the ocean but are also among the most vulnerable to ocean acidification
(OA) due to naturally high background concentrations of CO2. Yet our ability to predict how these ecosystems will respond to additional CO2
resulting from anthropogenic emissions is poor. To help address this uncertainty, researchers perform manipulative experiments where biological
responses are evaluated across different CO2 partial pressure (pCO2) levels. In upwelling systems, however, contemporary carbonate chemistry
variability remains only partly characterized and patterns of co-variation with other biologically important variables such as temperature and
oxygen are just beginning to be explored in the context of OA experimental design. If co-variation among variables is prevalent, researchers risk
performing OA experiments with control conditions that are not experienced by the focal species, potentially diminishing the ecological relevance
of the experiment. Here, we synthesized a large carbonate chemistry dataset that consists of carbonate chemistry, temperature, and oxygen measurements from multiple moorings and ship-based sampling campaigns from the California Current Ecosystem (CCE), and includes fjord and tidal
estuaries and open coastal waters. We evaluated patterns of pCO2 variability and highlight important co-variation between pCO2, temperature, and
oxygen. We subsequently compared environmental pCO2 – temperature measurements with conditions maintained in OA experiments that used
organisms from the CCE. By drawing such comparisons, researchers can gain insight into the ecological relevance of previously published OA experiments, but also identify species or life history stages that may already be influenced by contemporary carbonate chemistry conditions. We illustrate
the implications co-variation among environmental variables can have for the interpretation of OA experimental results and suggest an approach
for designing experiments with pCO2 levels that better reflect OA hypotheses while simultaneously recognizing natural co-variation with other
biologically relevant variables.
Keywords: California Current, climate change, hypoxia, multistressor experiment, pH.
Published by Oxford University Press on behalf of International Council for the Exploration of the Sea 2015. This work is written by (a) US
Government employee(s) and is in the public domain in the US.
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Interpretation and design of ocean acidification experiments
in upwelling systems in the context of carbonate chemistry
co-variation with temperature and oxygen
Ocean acidification and experimental design
Introduction
conditions may also naturally co-vary with other biologically relevant variables including temperature and O2 over multiple spatial
and temporal scales (Reum et al., 2014). This may have important
implications for the design and interpretation of ocean acidification
(OA) experiments because of the potential for non-additive interactions between carbonate chemistry, temperature, and O2 on organismal performance and ecological interactions (Wernberg et al., 2012,
Harvey et al., 2013; Koch et al., 2013; Kroeker et al., 2013). For many
organisms, aerobic capacity and metabolic scope (the amount of
energy that can be allocated to activities beyond those required for
basic existence) may be influenced strongly by temperature, and
adversely impacted by reductions in ambient oxygen availability or
increases in CO2 concentrations (Pörtner, 2010, 2012).
Given the combined effects that temperature and environmental
O2 and pCO2 have on organismal physiology and experimental evidence indicating non-additive interactions on response variables
related to fitness and ecosystem function (Pörtner and Farrell,
2008; Pörtner, 2010; Harvey et al., 2013; Kroeker et al., 2013), knowledge of their co-variability is essential for designing OA experiments
that adequately characterize biological performance under contemporary relative to future acidified conditions. Yet for workers
focused on laboratory OA experiments, these relationships are
rarely incorporated into experimental designs. This poses important potential drawbacks. Foremost, if carbonate chemistry strongly
covaries with temperature or O2, researchers risk running experiments with control-water characteristics that are atypical of the
habitat to which a focal organism/life stage may have acclimated
or adapted to. Recently, pCO2 was shown to range widely (200–
2500 matm) and co-vary with temperature and O2 in a fjord
located in the northeast Pacific (Reum et al., 2014). In that system,
the strength and direction of the relationships changed with
season, but also between subregions that differed in terms of vertical
mixing. Consequently, within a given season and region, organisms
occurring in low pCO2 waters experience temperatures and O2 levels
that differed from those experienced by organisms that occupy high
pCO2 waters, and this has direct implications for how ecologically
relevant OA experiments should be designed (Reum et al., 2014).
Here, we expand on the topic and evaluate the potential importance of this issue for upwelling systems. To do so, we have assembled
a large dataset of carbonate chemistry measurements from a variety
of habitats throughout the California Current Ecosystem (CCE), a
major eastern boundary upwelling system that supports highly productive foodwebs. Along the coast, equatorward winds during
spring and summer drive surface flow offshore in an Ekman layer,
leading to the upwelling of cold, salty, O2-poor, and nutrient- and
CO2-rich subsurface water. In fall and winter, the wind direction
reverses resulting in downwelling and shoreward advection of
oceanic waters which are relatively warm, fresh, O2-saturated,
nutrient-deplete, and near air–sea CO2 equilibrium (Evans et al.,
2011; Harris et al., 2013). The dataset presented here consists of
pCO2 measurements from multiple moorings and ship-based sampling campaigns that collectively span 148 of latitude, and includes
data from estuary and open coastal water habitats. Although the
extent of data is constrained in either time or space for any single
sampling campaign, the synthesis of many datasets offers an overview of the potential range of pCO2, temperature, and O2 conditions
experienced by organisms from this region. Subsequently, we compared environmental pCO2 and temperature measurements to conditions in OA experiments performed on organisms obtained from
populations that reside within the CCE. In doing so, we place these
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Coastal upwelling systems located along the eastern boundary of
ocean basins are some of the most productive ecosystems in the
ocean but are also among the most vulnerable to OA (Feely et al.,
2008; Gruber et al., 2012). The net transport of deep, nutrient-rich
waters to the sunlit surface by upwelling-favourable winds promotes
high rates of primary production which in turn supports productive
foodwebs and major fisheries (Fréon et al., 2009). However, subsurface and newly upwelled waters naturally exhibit low O2 and high
CO2 concentrations due to the remineralization of organic material
exported from surface layers. Consequently, they have a reduced
capacity to buffer against changes in carbonate chemistry resulting
from ocean uptake of anthropogenic CO2 relative to open-ocean
surface waters (Feely et al., 2008; Fassbender et al., 2011; Harris
et al., 2013). In eastern Pacific systems such as the California
Current, the CO2 burden and O2 drawdown due to respiration are
high because the source waters transported to upwelling centres
along the coast have been isolated from the surface for a few
decades (Feely et al., 2008; Hauri et al., 2009). In the California
Current, anthropogenic CO2 has already lowered pH by 0.1,
causing the depth of undersaturation with respect to aragonite to
shoal and expanding the spatial extent of undersaturated surface
waters (Feely et al., 2008; Gruber et al., 2012; Harris et al., 2013).
In only a few more decades, models suggest that the depth of undersaturation may shoal into the upper 75 m of the water column in
some regions year-round (Gruber et al., 2012; Hauri et al., 2013).
Given the economic, ecological, and biogeochemical importance
of eastern boundary upwelling regions, understanding how
species that compose these ecosystems will respond to OA has
emerged as a high research priority (Fabry et al., 2008; Gruber,
2011; Doney et al., 2012).
To evaluate the sensitivity of species to OA, researchers commonly rely on manipulative experiments where organisms are exposed to
different carbonate chemistry conditions. Typically, experiments
include “control” conditions that attempt to simulate contemporary or preindustrial CO2 concentrations and “acidified” treatments
that correspond to potential future CO2 uptake by the oceans. For
studies focused on organisms from low productivity, open-ocean
surface waters researchers can rely on IPCC scenarios of atmospheric pCO2 concentrations to identify potential carbonate chemistry
treatments because assumptions of air–sea pCO2 equilibrium are
often nearly met (Barry et al., 2010; Orr, 2011). However, in upwelling systems, CO2 levels are more variable relative to open ocean
waters due to the outcropping of high-CO2 subsurface waters and
high rates of primary production and respiration that strongly
modulate seawater carbonate chemistry (Hales et al., 2005; Feely
et al., 2008; Borges and Abril, 2011; Fassbender et al., 2011).
Consequently, OA experiments that use organisms from these habitats and that rely on IPCC future atmospheric CO2 scenarios to
devise control and acidified seawater treatments may inadequately
replicate contemporary carbonate chemistry or include treatments
that fail to reflect realistic future OA hypotheses (Barry et al.,
2010; Andersson and Mackenzie, 2012; McElhany and Busch, 2012).
Recognition of the importance of including environmentally
relevant pCO2 levels in OA experiments has grown considerably,
and has led to the use of seawater chemistry monitoring programmes to inform treatment design in several recent OA studies
(Hofmann et al., 2014). Less appreciated from an experimental perspective, however, is the possibility that carbonate chemistry
583
584
experiments in a larger environmental context and draw on observations that may help guide OA researchers in considering experimental designs that are more appropriate for organisms that
occur in the CCE and similar coastal systems.
Material and methods
Carbonate chemistry data
Figure 1. Map of coordinates where (a) environmental carbonate
chemistry data were obtained from the northern and central CCE from
moorings and ship-based underway and discrete samples, (b) the
locations where organisms (or their broodstock) included in published
OA experiments were collected. In (a), samples coded as Puget Sound
also include measurements from the adjoining Strait of Juan de Fuca.
For additional details on environmental carbonate chemistry datasets,
see Supplementary Table S1.
survey, which sampled waters along the continental shelf of western
North America; mooring pCO2 time-series of surface waters from
an open coast location (Newport, Oregon), and a shallow tidal
estuary (Netarts Bay, Oregon); and published carbonate chemistry
data from late summer and fall surveys in a large fjord estuary
complex (Strait of Juan de Fuca and Puget Sound, Washington;
Supplementary Table S1). Further details of the survey, sampling
dates, sampling method, and habitats from which data were obtained
are provided in Supplementary Table S1. Although the dataset does
not include all habitat types that occur within the CCE, we believe
these data are sufficiently representative enough to offer an overview
of key patterns of carbonate chemistry, temperature, and O2 covariation. For the purposes of the present study, we included all measurements extending up to 200 km from the coast.
For our analysis, we focused on pCO2 rather than other parameters of the carbonate system because pCO2 is directly changed
by anthropogenic CO2 emissions and is the most commonly used
treatment variable in species-exposure OA experiments. Further,
direct measurements of pCO2 were more widely available than
other parameters in the CDIAC and SOCAT databases, and when
not available were readily estimated from other measured parameters of the carbonate system (see Supplementary Table S1 for
details on estimation methods). We recognize that organisms may
potentially be more sensitive to other variables of the carbonate
system such as pH or to changes in the calcium carbonate saturation
state of seawater (Barton et al., 2012; Waldbusser and Salisbury,
2013) and that waters with similar pCO2 values may differ with
respect to aragonite or calcite saturation states if total alkalinities
differ. However, pCO2 is routinely reported in OA studies and is
the parameter that allowed us to best standardize comparisons
among experiments and between experiments and environmental
carbonate chemistry measurements. Further, the focus on pCO2
prevented the use of some carbonate chemistry datasets such as
those including information only on pH (e.g. Hofmann et al.,
2011). We limited our analysis to pCO2 measurements taken from
the top 50 m of the water column because the species and life
history stages for which OA experiments have been performed
from the northern and central CCE typically occur in waters
within this depth range, although individuals of some populations
may occur for periods below this depth.
We evaluated separate relationships between pCO2 and temperature and pCO2 and O2 within summer upwelling and winter downwelling periods. Co-variation patterns for pCO2 and temperature were
examined for data pooled from shipboard underway and discrete
sample measurements collected along the West Coast continental
shelf. In addition, we also examined co-variation patterns for Puget
Sound and the Strait of Juan de Fuca discrete sample measurements
(hereafter referred to as “Puget Sound”). A subset of these data were
examined in an earlier study (Reum et al., 2014), but we include
them here for completeness and to facilitate comparisons among habitats represented within the CCE. We chose to present co-variation patterns for Puget Sound separately from the West Coast because Puget
Sound exhibits slow exchange with open coastal waters and high
rates of primary productivity and respiration (Feely et al., 2010). The
resulting pCO2 –temperature relationships therefore likely differ considerably from patterns observed in shelf waters. For the time-series
data, we estimated pCO2 –temperature relationships for upwelling
and downwelling seasons, but examined co-variation patterns
separately for each mooring to evaluate site-level differences.
To evaluate pCO2 and O2 relationships and facilitate comparisons
across survey types and seasons, we converted all O2 concentration
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Our analysis focused on coastal waters within the domain of the northern and central CCE (Figure 1). The region extends from northern
Vancouver Island, British Columbia (508N) to Point Conception,
California (348N), and is typified by distinct wind-driven upwelling
(May –October) and downwelling (November– April) oceanographic seasons (Checkley and Barth, 2009). The region includes
large expanses of open coastal habitat and many inlets and estuaries
that range considerably in terms of surface area, depth, substrate,
and connectivity with the open ocean (Hickey and Banas, 2003).
To obtain an overview of the potential range of temperatures and
carbonate chemistry conditions, we retrieved all publicly available
records of measurements sampled within our region of interest
from the Carbon Dioxide Information Analysis Center (CDIAC)
and Surface Ocean CO2 Atlas (SOCAT, Bakker et al., 2013). The
dataset includes carbonate chemistry measurements from discrete
water samples collected at depth, shipboard measurements of surface
water pCO2, and mooring pCO2 time-series of surface waters off
the coast of California and Washington (Supplementary Table
S1). In addition to CDIAC and SOCAT data, we also included unpublished carbonate chemistry data obtained from the 2011 and
2012 NOAA Ocean Acidification Programme West Coast Cruise
J. C. P. Reum et al.
585
Ocean acidification and experimental design
measurements to micromole per kilogramme. As with the pCO2 and
temperature data, we examined co-variation within data pooled from
shipboard West Coast measurements, Puget Sound, and each
mooring and by season. To improve assumptions of normality in
the residual error structure for the pCO2 –temperature and pCO2 –
O2 relationships, we log10-transformed pCO2 values before estimating
linear relationships using least-squares regression. For presentation
purposes, the liner relationships were back-transformed to the original
pCO2 scale. Our goal was to estimate overall mean relationships within
seasons and survey types to facilitate visual inspection and to evaluate
co-variation patterns in an exploratory manner. We therefore did not
test for significant differences between seasons or survey types. All
linear relationships were fitted using the R version 2.11 statistical software package (R Development Core Team, 2011).
We searched the published literature for OA experiments that
included organisms obtained directly from habitats within the northern and central CCE or that originated from broodstock collected
from the region. To do so, we searched Google Scholar, ISI databases
(Web of Science, Current Contents), and references included in recently published reviews and meta-analyses that address OA
(Dupont et al., 2010; Wernberg et al., 2012; Harvey et al., 2013;
Kroeker et al., 2013; Wittmann and Pörtner, 2013). We used the
search terms “ocean acidification”, “carbon dioxide”, “experiment”,
and “manipulation”. We included all studies published through
15 October 2013 that were found under these search criteria.
From each study, we retrieved information on the collection site
of the organism (or their broodstock) and the temperatures and
pCO2 levels at which the experiment was performed. For nearly all
studies, experimental O2 concentrations were not reported nor
could saturation conditions be safely assumed. We therefore
focused our comparison of environmental and experimental conditions to pCO2 and temperature. Following the authors’ interpretation of the results, we recorded whether the dependent biological
variables differed significantly in a positive or negative direction
or showed no significant difference relative to the control pCO2
level specified by the author. If authors did not explicitly designate
a control pCO2 level in their study, we considered treatments with
pCO2 levels closest to present-day air pCO2 levels (400 matm) as
the control to facilitate comparisons across studies. When more
than one response variable was tested in an experiment, we coded
the net outcome of the experiment at a given treatment level
based on the result of the variable most sensitive to pCO2.
From the outset, we recognized that the species and life history
stages for which published OA experimental data were available
held distributions that spanned large sections of the northern and
central CCE or were planktonic with high dispersal potential.
Further, the exact dispersal patterns of many of these species are
not well understood. Although larvae of some species may be functionally limited to a subset of the CCE system (e.g. some species may
occur primarily in bays or estuaries), “spillage” into adjoining habitats and waters through advective processes is also probable. We
therefore used the full dataset of field pCO2 and temperature measurements to demarcate the potential pCO2 – temperature space
organisms may encounter. We acknowledge that the actual pCO2
levels and temperatures experienced by organisms will differ for
populations across locations, seasons, and due to possible interannual variation in upwelling and climate forcing. Our main intention,
however, was to draw comparisons between the conditions maintained in OA experiments and the full range of environmental
Results
pCO2 , temperature, and O2 co-variation
Temperature and pCO2 values in the upper 50 m of the CCE spanned
6–198C and 100–1500 matm, respectively, based on the pooled shipboard underway and discrete water sample measurements along the
open coast. pCO2 values tended to increase with decreasing water temperature during summer upwelling months (Figure 2a). In Puget
Sound, a similar but steeper relationship was apparent relative to the
open coastal waters, reflecting CO2-enriched waters (Figure 2a).
Along the open coast, the range of pCO2 values was also wider at
cool relative to warm temperatures. For instance, pCO2 values at
98C ranged from 320 to 1400 matm, while at 168C, the range extended
from 130 to 420 matm. In Puget Sound, the pCO2 range was also wider
at cooler temperatures (Figure 2a).
Although fewer measurements were available during winter in
general (Supplementary Table S1), the data showed weak co-variation
between pCO2 and temperature in open coastal waters and the
overall range of pCO2 values and temperatures narrowed relative
to summer (Figure 2b). This is due to the relative absence of the
cold, high- pCO2 upwelled source waters. In contrast, winter
pCO2 and temperature values in Puget Sound positively covaried,
and the range of pCO2 values and temperatures also narrowed relative to summer (Figure 2b).
Time-series data collected from moored platforms in open
coastal waters from four locations also indicated co-variation
between pCO2 and temperature (Figure 3a –c). During summer upwelling months, pCO2 again generally declined with increasing temperature (Figure 3a –c). The range of pCO2 levels among stations,
however, differed substantially. Time-series data collected at moorings in shelf waters off Washington and near Point Conception, CA.
(Figure 3a and b) ranged in pCO2 from 200 to 600 and 300 to
600 matm, respectively, while off the coast of Oregon the range
spanned 200–1100 matm (Figure 3c). The differences among stations reflect considerable spatial variation in the supply of newly
upwelled waters.
Time-series data collected from Netarts Bay, a shallow tidal estuary
in northern Oregon, indicated summer range in pCO2 values was also
considerable, spanning 300–800 matm and reflected high rates of
primary production and respiration (Figure 3d). The overall relationship between pCO2 and temperature, however, was also negative. In
winter, co-variation between pCO2 and temperature at all mooring
stations was weaker relative to summer but variable across stations
(Figure 3d). In addition, the range of temperatures and pCO2
values were generally lower in winter relative to summer in two of
the five stations (Figure 3c and d).
Measurements of O2 were available for a subset of pCO2 records
included in the complete environmental pCO2 and temperature
dataset (Supplementary Table S1). Forall survey types, mooring timeseries, and seasons, pCO2 levels decreased with increasing O2 concentration (Figure 4). For most moorings on the shelf, O2 ranged from
230 to 350 mmol kg21 (both seasons combined). Summer O2 levels
at Newport Oregon, however, ranged from 120 to 350 mmol kg21
(Figure 4). Summer O2 measurements from discrete samples from
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OA experimental studies
pCO2 and temperature values based on empirical observations. In
the absence of detailed information on the fine-scale distribution
and movement patterns of most species and life history stages,
and environmental pCO2 –temperature measurements of matching
resolution, comparisons at finer spatio-temporal scales were not
possible.
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J. C. P. Reum et al.
Puget Sound and the West Coast ranged more widely, from 40 to
400 mmol kg21, where concentrations below 60 mmol kg21 reflect
hypoxic conditions (Figure 4). The general negative relationship
between pCO2 and O2 corresponds to the well-understood effects
of aerobic respiration and photosynthesis in marine ecosystems.
When aerobic respiration dominates, CO2 is remineralized and O2
levels are drawn down, while the reverse occurs when photosynthesis
dominates.
OA studies
In total, 26 experiments from 22 published OA studies included
organisms (or broodstock) from the northern and central CCE
(Figure 1b) and provided sufficient information on treatment
water conditions to compare with the CCE pCO2 – temperature
space defined by combining all environmental datasets (Table 1).
In terms of taxonomic diversity, 50, 35, and 15% of experiments
evaluated responses in echinoderms, molluscs, and teleosts, respectively. However, 35% of all experiments focused on responses in a
single species, the echinoderm Strongylocentrotus purpuratus
(Table 1). Of the experiments, 81% examined gamete, egg, or
larval performance (survival, growth, calcification rates), while
the remaining studies evaluated performance metrics (growth, development, or calcification rates) in juvenile or adult life history
stages (Table 1). One study measured genetic diversity in echinoderm larvae after exposure to different pCO2 treatments to evaluate
evolutionary potential.
To facilitate visual inspection and comparison of experimental
pCO2 and temperature treatments with the pCO2 –temperature
space defined by the complete set of environmental measurements
in our dataset, we examined echinoderm and non-echinoderm
studies separately (Figure 5). Overall, OA experiments included
pCO2 treatment levels that extended from 200 to 4000 matm; 42%
of experiments included a single elevated pCO2 treatment in addition to a control at a given temperature, 38% considered two
different elevated pCO2 treatments, and 19% included three or
more elevated treatments (Figure 5). In total, 72% of experiments
included at least one control and one elevated pCO2 treatment
that occurred within the observed range of pCO2 and temperature
values in the CCE. Of those, 63% (10 out of 16) observed negative
biological effects, 18% observed positive effects, and 18% observed
no effect relative to control pCO2 levels. Only two studies included
pCO2 treatments below approximate present-day atmospheric
levels (400 matm; Figure 5).
Compared with the pCO2 – temperature space defined by the
complete set of environmental measurements in our dataset, five
experiments were performed at temperatures that matched or
exceeded the warmest observed values (198C; Figure 5). These
included three experiments on the early life history stages of the
native Olympia oyster, an experiment on sand dollar larvae, and
an experiment on the non-native Pacific oyster which is routinely
reared at 208C to optimize survival under commercial hatchery
conditions. One experiment included a 2.18C treatment; though
this temperature was meant to simulate cool conditions in
Alaskan waters, the source stock was collected near Puget Sound
(Table 1).
A review of each study indicated that Intergovernmental Panel
on Climate Change (IPCC) estimates of future global surface
ocean mean pCO2 levels were used as the sole rationale for selecting
OA treatments in 45% of studies, while 31% cited a combination of
regional modelling studies, local field measurements, and IPCC estimates to support their choice of experimental pCO2 treatment
levels. Of the remaining studies, 13% provided no rationale for
their choice of pCO2 treatment levels, one based the high pCO2 treatment level on observations of contemporary upwelling conditions,
and one noted natural high carbonate chemistry variability in
coastal upwelling systems which necessitated the need to test biological responses to a wide range of pCO2 levels. In terms of temperature, 80% of studies did not provide a rationale for their choice of
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Figure 2. (a) Relationship between pCO2 and temperature in the top 50 m of the water column in the northern and central CCE during summer
upwelling season which approximately spans May through October and (b) winter (November through April) when downwelling-favourable winds
predominate. All non-time-series data are displayed. Measurements of pCO2 from cool waters in Puget Sound are elevated relative to other regions
sampled in the CCE. Regression lines are overlaid to aid evaluation of patterns. Lines labelled P and C denote relationships for Puget Sound and open
coastal locations, respectively. For reference, approximate present-day pCO2 levels (390 matm) are indicated by the dashed horizontal line.
Ocean acidification and experimental design
587
experimental levels. The remaining studies cited similarity to local
field conditions as a rationale for selecting temperatures. Of the
two studies that performed experiments that included crossed temperature and pCO2 treatments meant to correspond to conditions in
the CCE, the warmer treatments were chosen in reference to IPCC
global average temperature increase projections.
Discussion
Single-species response experiments have offered important initial
evidence that some species and life history stages may be adversely
impacted by OA (Kroeker et al., 2013; Wittmann and Pörtner,
2013), but there is a pressing need for the development of experiments that include more appropriate reference treatments that
reflect pCO2 levels species have acclimated or adapted to and
acidified treatments that more closely reflect natural heterogeneity
in carbonate chemistry (Barry et al., 2010; Andersson and
Mackenzie, 2012; McElhany and Busch, 2012; Waldbusser and
Salisbury, 2013). Our analysis shows that an additional source of
concern is natural co-variation between carbonate chemistry and
other biologically relevant variables including temperature and
O2. These findings have direct consequences for designing experiments that aim to include control treatments that are similar to
natural water conditions and for selecting elevated pCO2 treatment
levels that more closely correspond to OA hypotheses. Further, by
placing the findings of published OA experiments into a larger
environmental context, we gain information on the ecological
relevance of experimental water conditions and insight into the
potential sensitivity of some species and life stages to carbonate
chemistry conditions that already occur in the CCE.
Although researchers increasingly recognize the importance of
basing experimental pCO2 levels on in situ carbonate chemistry observations (Yu et al., 2011; Evans et al., 2013; Hofmann et al., 2014), the
implications of natural co-variationwith temperature or O2 to experimental design and inference are only beginning to be explored. Given
experimental evidence and theoretical expectations of interactive or
synergistic effects between pCO2, temperature, and O2 on organisms
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Figure 3. Relationship between pCO2 and temperature of surface waters measured during summer (light grey open circle symbols) and winter
(dark grey) at open coastal moorings in (a) Washington, (b) California, and (c) Oregon, and (d) a tidal estuary in Oregon. Regression lines are overlaid
and labelled S and W to indicate summer or winter, respectively. Dashed line convex hulls demarcate data ranges where seasons overlap. For
reference, approximate present-day pCO2 levels (390 matm) are indicated by the dashed horizontal line.
588
(Pörtner, 2010, 2012; Harvey et al., 2013), we suggest that failure to
account for natural co-variation among these variables in habitats
from the CCE may lead to results with diminished relevance for
making predictions. To illustrate this point, we present an example
multistressor experimental scheme typical of published OA experiments in which temperature (three levels at 8, 12, and 168C) is
crossed with two pCO2 levels that correspond to approximate global
surface ocean present-day (400 matm) and future (800 matm) conditions (Figure 6a). Under the conventional method, treatments are
fully orthogonal which permits estimates of the effect sizes of the individual predictor variables and of their interaction on the response variable. The method holds merit as a tool for comparing the relative
influence that each predictor has on the response variable, is widely
applied in multistressor experiments, and facilitates the development
of mechanistic models. However, if a goal of a study is to evaluate
the potential sensitivity of organisms to future OA as is often the
case, the design may be inadequate, given natural pCO2 –temperature
co-variation within different habitats and water masses. For example,
assuming an organism of interest occurs in shelf waters off Oregon
during summer upwelling months (e.g. a pelagic larval invertebrate),
the assumption that 800 matm corresponds to a future OA prediction
across all temperatures is not accurate. At the Newport, Oregon,
mooring pCO2 levels of 800 matm already occur at 88C under presentday conditions and “control” 400 matm waters do not (Figure 6a). At
temperatures above 138C, the mean pCO2 values approach air–sea
equilibrium conditions. We do not doubt that simple crossed experimental designs will provide information on the interactive effects of
pCO2 and temperature, but we do question the efficacy of the design
for testing OA hypotheses on future ecological response to OA, given
naturally occurring pCO2 –temperature relationships and the wide
range of both variables in the CCE.
In light of potential co-variation between carbonate chemistry
and other important environmental variables, how should
researchers select pCO2 treatments that correspond to OA hypotheses? As a starting point, we recommend that OA experimental
designs include multiple controls that reflect the span of pCO2
levels and temperatures likely to be experienced by the organism
under study (Figure 5b). To design pCO2 treatments that represent
future OA scenarios in productive coastal systems, we suggest that
researchers focus on changes in the anthropogenic contribution
to in situ dissolved inorganic carbon, DIC (e.g. Feely et al., 2008,
2010; Barry et al., 2010; Melzner et al., 2012; Shaw et al., 2013). At
the Newport, Oregon, mooring, newly upwelled waters exhibit
pCO2 values that are elevated relative to air–sea equilibrium conditions due to the remineralization of organic material before
surfacing (Evans et al., 2011). However, after surfacing CO2 concentrations can be drawn down rapidly by photosynthesis (Hales et al.,
2005; Hales et al., 2006; Evans et al., 2011), often at rates that typically
far exceed CO2 equilibration times across the air–sea interface (e.g.
van Geen et al., 2000; Fassbender et al., 2011). Consequently, the anthropogenic CO2 burden of upwelled waters is primarily acquired
when they were last in contact with the atmosphere and before DIC
changes due to biological processes post-surfacing. In our example,
pCO2 treatments reflecting future OA hypotheses could be obtained
by increasing in situ DIC concentrations by an increment (DDIC)
expected under a given CO2 emissions scenario. The future DIC estimate (DDIC + in situ DIC), along with a second parameter from the
carbonate system, could then be used to recalculate the carbonate
system to estimate treatment pCO2 levels.
Because the Newport, Oregon, time-series consists only of pCO2,
we first needed to estimate in situ DIC. To do so, we estimated total
alkalinity (TA) from salinity measurements using a linear model
parameterized with data from the CCE (Gray et al., 2011). The relationship has relatively low residual error (approximately
+20 mmol kg21) and was shown previously to adequately predict
TA for the purposes of estimating the carbonate system when
using pCO2 as the second parameter (Harris et al., 2013). We used
the estimated TA and in situ pCO2, salinity, and temperature measurements to solve the carbonate system and calculate in situ DIC.
To estimate DDIC, we solved the carbonate system based on estimates of TA, and in situ temperature, and salinity, but assuming seawater equilibrium with average atmospheric pCO2 levels during the
period of the moored observations (390 matm; Harris et al., 2013)
and those predicted under an emissions scenario for the year 2100
(788 matm; Intergovernmental Panel on Climate Change IS92a
“continually increasing” emissions scenario). The present-day
air– sea equilibrium DIC estimate was subtracted from the future
equilibrium estimate to obtain DDIC. Although the same present
day and future pCO2 levels were used to calculate DDIC for all
samples, DDIC values differ among samples because estimates of
DIC at air–sea pCO2 equilibrium vary based on the TA, temperature, and salinity of the sample. Across all water samples, DDIC
spanned 75 –105 mmol kg21 depending on the TA and temperature of samples. We then obtained future pCO2 estimates by solving
the carbonate system using the estimated TA and in situ DIC +
DDIC values (Figure 6b). Versions of the method have been
described previously (e.g. Barry et al., 2010) and can be used to
estimate preindustrial carbonate chemistry conditions (e.g. Feely
et al., 2008; Harris et al., 2013).
Under this approach, and assuming the same number of treatments is used as depicted in Figure 6a, the effects of temperature
and pCO2 can no longer be separated because orthogonality in the
design is lost (Figure 6b). However, a more realistic set of control
treatments are included that offer a firmer basis for drawing
Downloaded from http://icesjms.oxfordjournals.org/ at Oregon State University on April 11, 2016
Figure 4. Relationships between pCO2 and O2 in the top 50 m of the
water column during summer upwelling (May through October) and
winter downwelling seasons (November through April). Regression lines
are overlaid for summer and winter; lines labelled M, P, and C correspond
to mooring, Puget Sound, and open coast datasets, respectively. For
reference, approximate present-day pCO2 levels (390 matm) are
indicated by the dashed horizontal line, and the hypoxia threshold
(60 mmol kg21) is indicated with a dashed vertical line.
J. C. P. Reum et al.
Study
number
1
Reference
Gooding et al. (2009)
Collection site
Jaricho Beach, BC
2
Reuter et al. (2011)
Barkley Sound, BC
3
Nienhuis et al. (2010)
Barkley Sound, BC
4
Sunday et al. (2011)
Barkley Sound, BC
4
Sunday et al. (2011)
Barkley Sound, BC
5
Crim et al. (2011)
Barkley Sound, BC
6
Chan et al. (2011)
Orcas Island, WA
7
Timmins-Schiffman
San Juan Island,
et al. (2012)
WA
O’Donnell et al. (2013) San Juan Island,
WA
8
Species
(common name)
Pisaster ochraceus
(purple sea star)
Strongylocentrotus
franciscanus
(red sea urchin)
Nucella lamellose
(Frilled dogwinkle)
Strongylocentrotus
franciscanus
(red sea urchin)
Mytilus trossulus
(bay mussel)
Haliotis kamtschatkana
(northern abalone)
Dendraster excentricus
(Pacific sand dollar)
Crassostrea gigas
(Pacific oyster)
Mytilus trossulus
(bay mussel)
Life stage
Juvenile
Duration
(days,
hours)
70 d
Control
pCO2
T (88 C) (matm)
12, 15 380
Treatment pCO2
(matm)
780
Outcome relative to control
Increased growth rate, reduced calcified
mass; feeding and growth increased with
temperature
Decreased range of sperm concentrations
over which high fertilization success was
likely
Enhanced shell dissolution
Sperm/eggs
1h
10.2
400
800, 1800
Adults
6d
9.0
380
780, 1585
Larvae
1d
12.0
400
1000
Reduced larval size, large variation among
families
Larvae
1d
12.0
400
1000
Larvae
8d
12.0
400
800, 1800
Larvae
10 d
20.0
380
1000
Larvae
3d
20.0
468
847, 1065
Adults
20 d
10.4
432
42 d
8.8
414
299, 575, 736, 980,
1057, 1180,
1322, 1498
478, 815, 1805
Reduced larval size, small variation among
families
Reduced survival, increased abnormalities in
shell structure, reduced size in
normal-shelled larvae
Reduced body size and stomachs; no effect
on swimming speed
Increased calcification at day 1, but smaller
average size on day 3
Weaker, less flexible byssal threads at
treatments above 1200 matm
196 d
8.3
596
828, 1285, 2894
196 d
2.4
386
225, 643, 1543
35 d
8.1
442
296, 871, 1844
Larvae
96 h
12.8
435
813, 1255
Larvae
17 d
14.1
428
897
9
Hurst et al. (2012)
Port Townsend,
WA
Theragra chalcogramma Yearlings
(walleye pollock)
9
Hurst et al. (2012)
9
Hurst et al. (2012)
10
Hurst et al. (2013)
Port Townsend,
WA
Port Townsend,
WA
Port Townsend,
WA
Theragra chalcogramma Sub-yearlings
(walleye pollock)
warm
Theragra chalcogramma Sub-yearlings
(walleye pollock)
cool
Theragra chalcogramma Eggs/larvae
(walleye pollock)
11
Evans et al. (2013)
Fogarty Creek, OR
12
Pespeni et al. (2013)
OR to CA., various
locations
Strongylocentrotus
purpuratus
(purple sea urchin)
Strongylocentrotus
purpuratus
(purple sea urchin)
Ocean acidification and experimental design
Table 1. Brief summary of experimental outcomes of reviewed OA studies that focus on organisms (or their broodstock) collected from the CCE.
No effect on growth but increase in otolith
deposition rate, biological implication is
unclear
Increase growth at pCO2 higher than 900
matm; no change in condition factor
No difference in growth or condition factor
across treatments
No difference in egg hatch rate, size, or
survival, but longer time till hatching;
authors think elevated pCO2 treatments
have minor effect
Transcriptome up-regulation of genes
related to calcification at intermediate
pCO2, no change at high level
Reduction in larval body length, change in
allele frequency, no change in timing of
settlement or competence to
metamorphose
589
Continued
Downloaded from http://icesjms.oxfordjournals.org/ at Oregon State University on April 11, 2016
590
Table 1. Continued
14
15
16
17
18
Species
Reference
Collection site
(common name)
LaVigne et al. (2012)
OR to CA, various Strongylocentrotus
locations
purpuratus
(purple sea urchin)
Kelly et al. (2013)
Northern and
Strongylocentrotus
southern CA,
purpuratus
various locations
(purple sea urchin)
Place and Smith
Bodega Bay, CA
Strongylocentrotus
(2012)
purpuratus
(purple sea urchin)
Hettinger et al. (2012) Tomales Bay, CA
Ostreola lurida
(Olympia oyster)
Hettinger et al. (2013a) Tomales Bay, CA
Ostreola lurida
(Olympia oyster)
Hettinger et al.
Tomales Bay, CA
Ostreola lurida
(2013b)
(Olympia oyster)
19
Gaylord et al. (2011)
Tomales Bay, CA
20
Padilla-Gamiño et al.
(2013)
Santa Barbara, CA
21
Matson et al. (2012)
Santa Barbara, CA
22
Yu et al. (2011)
Santa Barbara, CA
22
Yu et al. (2011)
Santa Barbara, CA
Mytilus californianus
(California mussel)
Strongylocentrotus
purpuratus
(purple sea urchin)
Strongylocentrotus
purpuratus
(purple sea urchin)
Strongylocentrotus
purpuratus
(purple sea urchin)
Strongylocentrotus
purpuratus
(purple sea urchin)
Life stage
Larvae
Duration
(days,
hours)
50 d
Control
pCO2
T (88 C) (matm)
14.1
490
Treatment pCO2
(matm)
1001
Larvae
5d
13.0
424
1210
Embryos
7d
15.1
378
1486, 4110
Outcome relative to control
No change Sr/Mg composition in spines;
though a difference was observed for a
Santa Barbara population of urchins
Reduced size in larvae from dams and sires
taken from northern and southern
locations; no difference in metabolism
No disruption to cell cycle in fertilized eggs
Larvae/
juveniles
Larvae
45 d
20.0
739
933, 1355
Reduced larval and juvenile growth
22 d
19.4
520
1075
Larvae/
juveniles
127 d
20.0
485
1060
Larvae
8d
15.4
380
540, 970
Reduced number of settlers; no difference in
shell size or larval dry weight
Reduced larval survival and growth; reduced
growth in juveniles outplanted to an
estuary
Thinner, weaker shells
Larvae
75 h
Larvae
13, 18 400
1100
Reduced body size and respiration rate;
difference in transcriptome observed
6d
15.6
365
1038, 1444
Larvae
6d
15.6
372
1057, 1469
Reduced arm length; no difference in
utilization of energy lipid reserves and
protein content remained unchanged
Slight reduction in size
Larvae
6d
15.6
432
1441
Slight reduction in size
J. C. P. Reum et al.
Downloaded from http://icesjms.oxfordjournals.org/ at Oregon State University on April 11, 2016
Study
number
13
Ocean acidification and experimental design
591
Figure 6. Schematic of potential experimental approaches to evaluate OA effects, given co-variation between pCO2 and temperature. To illustrate
the benefits and drawback of each approach, in situ pCO2 and temperature measurements from the N10 mooring near Newport, Oregon,
during summer upwelling season (2008) are depicted (grey, filled squares; bars indicate standard deviation). (a) A conventional temperature (three
levels: 8, 12, and 168C) by pCO2 experimental design in which control pCO2 values are based on approximate present-day global average
surface ocean pCO2 levels and the acidified treatments corresponding to IPCC emissions scenario IS92a projections for year 2100 (390 and
788 matm; open square and circle symbols, respectively). Arrows indicate statistical comparisons permitted by the design. (b) Experimental design
informed by in situ pCO2 and temperature measurements. Under this design, three controls are included to account for natural co-variation in
temperature and pCO2. Treatment levels that more closely correspond to an OA hypothesis were obtained by specifying an increase in DIC
attributed to anthropogenic CO2 emissions (see Discussion for details). The future DIC estimate and estimates of TA were used to recalculate the
carbonate system to obtain target pCO2 treatment levels. We calculated pCO2 using the R library “seacarb” (Lavigne and Gattuso, 2010) with
dissociation constants from Lueker et al. (2000).
inferences about future OA impacts at a given temperature. The
experimental design could be improved further by using O2
concentrations that currently occur at the three different pCO2 –
temperature controls.
The method we use to estimate future pCO2 requires several
important assumptions. First, the approach implicitly assumes
that TA, salinity, and temperature will remain unchanged and that
future difference in DIC between the observed in situ values and
Downloaded from http://icesjms.oxfordjournals.org/ at Oregon State University on April 11, 2016
Figure 5. Environmental pCO2 and temperature measurements (top 50 m) from the northern and central CCE and conditions maintained in OA
experiments performed on organisms from the region. Dark grey circles correspond to environmental measurements from Puget Sound,
Washington; light grey circles correspond to environmental measurements from all other regions. pCO2 treatment levels included in an individual
experiment at a given temperature are connected by solid black lines. Numbers denote study code (see Table 1). A convex hull (solid grey line)
demarcating the extent of all environmental pCO2 and temperature measurements is depicted to aid visual comparisons. For reference,
approximate present-day atmospheric pCO2 levels (390 matm) are indicated by the dashed horizontal line.
592
storms or the advection of a different water mass into a region can
result in rapid change in water characteristics that may persist for
several days (Frieder et al., 2012). Over interannual time-scales,
large-scale climate phenomena like El Niño/Southern Oscillation
can influence upwelling patterns, coastal productivity, carbonate
chemistry (Chavez et al., 1999; Friederich et al., 2002), and thus potentially relationships among pCO2 and temperature and O2. To
date, studies evaluating patterns of co-variation between pCO2
and other biologically relevant variables over a range of temporal
scales are sparse, but the topic is an area of research we are currently
exploring. Incorporating both temporal dynamics and multiple
stressors in experimental systems is technically challenging, but
may be necessary for ecologically relevant predictions.
Conclusions
OA is expected to have far-reaching impacts on the structure and
function of marine ecosystems by altering biogeochemical processes
and the productivity and distribution of species (Fabry et al., 2008;
Doney et al., 2009, 2012; Mora et al., 2013; Waldbusser and
Salisbury, 2013). Our ability to predict the response of complex ecological systems to OA, however, remains limited and is highly constrained by major uncertainties in the response of species to both
direct (e.g. physiology, neurological impairment) and indirect
(e.g. trophic interactions) processes that may be vulnerable to OA
(Fabry et al., 2008; Hofmann et al., 2010). While authors have
noted the need for OA researchers to use pCO2 levels that correspond to ambient conditions a study species or life history stage is
likely to experience (Andersson and Mackenzie, 2012; McElhany
and Busch, 2012), patterns of co-variation with temperature and
O2 have yet to be incorporated into OA experimental designs
(Reum et al., 2014). As demonstrated here, this issue should be of
concern to researchers in upwelling systems and other coastal environments where water conditions are highly dynamic over a range of
spatial and temporal scales and where co-variation between pCO2,
temperature, and O2 are generally expected. Because inferences on
the potential response of organisms to future conditions are necessarily premised on the notion that experimental controls reflect
present-day conditions, we strongly recommend researchers consider how pCO2 naturally varies with other biologically important
variables in their experimental designs. Importantly, the studies
we reviewed for the CCE indicate that several species may be sensitive to carbonate chemistry conditions that already occur, and
suggest that present-day variability in carbonate chemistry may be
more important to contemporary ecological patterns than previously thought. With the continued collection of high-quality carbonate chemistry measurements and their archival on freely
accessible databases, analyses like the one we present here for the
CCE may yield further insight into the relevance of carbonate chemistry variability to contemporary ecological processes as well as
guide OA experimental design in other marine systems.
Supplementary data
Supplementary material is available at the ICESJMS online version
of the manuscript.
Acknowledgements
The authors wish to thank many research technical staff in the laboratories that contributed data used in this manuscript, particularly the scientific and engineering staff in NOAA’s Pacific Marine
Environmental Laboratory (PMEL) carbon, CTD-O2, and engineering groups; UW’s Applied Physics Laboratory (UW/APL) and
Downloaded from http://icesjms.oxfordjournals.org/ at Oregon State University on April 11, 2016
those calculated assuming present-day air–sea equilibrium will
remain the same. Further, to estimate DDIC, we assumed that all
water properties measured at the time of sampling are the same as
when the water mass last approached air– sea equilibrium. If
waters were cooler at that time, this would result in a slight overestimate of DDIC of 1.6 mmol kg21 for each degree Celsius.
Following earlier studies, we also assumed that waters upwelled at
the mooring location possessed an anthropogenic CO2 burden
that approximated present-day atmospheric pCO2 conditions
(Harris et al., 2013). In other locations within the CCE, such
assumptions may not be justified because subsurface waters
upwelled on to the shelf may last have had contact with the
surface decades prior and therefore would contain less
anthropogenic CO2 (Feely et al., 2008; Harris et al., 2013).
The OA pCO2 –temperature relationship depicted in Figure 6b
corresponds to one simple hypothesis for how present-day carbonate chemistry conditions might change with a simple augmentation
of CO2. However, marine organisms will be subjected to multiple
stressors in the future, including warmer temperatures, lower
ambient O2 concentrations, cultural eutrophication, and pollution
(Boyd, 2011; Gruber, 2011; Doney et al., 2012). Treatments corresponding to an OA + warming hypothesis could be created by
adding to in situ measurements of both temperature and DIC and
recalculating the carbonate system to obtain appropriate estimates
of pCO2 (Melzner et al., 2012). If a hypoxia + OA treatment is
sought, any assumed reduction in O2 owing to aerobic respiration
necessarily corresponds to an increase in CO2 (Melzner et al.,
2012; Sunda and Cai, 2012). The corresponding increase in DIC
beyond that attributed to OA could be estimated based on the
molar ratio of O2 consumed to CO2 released in the respiratory consumption of organic matter (Sunda and Cai, 2012). We caution,
however, that the appropriateness of these simple methods for estimating treatment levels should be thoughtfully considered in the
light of the physical and biological attributes of the system under
study. Such simplifications do not take into account the indirect
and cascading impacts that changes in individual properties such
as temperature will have on ecosystem metabolism and thus the distribution and concentration of O2 and CO2 (e.g. Keeling et al., 2010;
Gruber, 2011; Doney et al., 2012), nor would they reflect potential
changes in large-scale circulation patterns or productivity regimes
which might fundamentally alter relationships between pCO2 and
other variables (e.g. Rykaczewski and Dunne, 2010). Despite these
uncertainties, developing experimental designs that include controls that reflect present-day pCO2 –temperature–O2 relationships
in upwelling systems should become the cornerstone of experiments
that aim to quantify the potential response of organism to future
predicted changes in their environment. The development of experimental systems that permit simultaneous control over pCO2,
temperature, and O2 conditions remains a technical challenge, but
a growing number of OA research facilities are acquiring the capacity
to do so (Bockmon et al., 2013).
Our compilation of environmental carbonate chemistry data is
meant to offer an initial overview of the ranges of pCO2, temperature, and O2 in the CCE and the extent to which their relationships
vary between seasons and regions. We examined patterns of covariation between pCO2 and temperature at coarse seasonal timescales, but note that co-variation patterns may differ depending
on temporal scale. On diel time-scales, solar heating and photosynthesis and respiration may drive strong cyclical patterns in temperature, pCO2, and O2 (Barton et al., 2012; Frieder et al., 2012;
Waldbusser and Salisbury, 2013), while abrupt events such as
J. C. P. Reum et al.
Ocean acidification and experimental design
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School of Oceanography; OSU’s College of Earth, Ocean, and
Atmospheric Sciences Ocean Ecology and Biogeochemistry division; and the Scripps Institution of Oceanography Ocean Time
Series Group for the tremendous effort involved in making these
datasets available. In addition, we thank the officers and crew on
all spatial surveys, underway observing ships, and mooring deployments for their able assistance during field operations. Funding for
JCPR was provided through a National Research Council
Fellowship. Funding for NOAA/PMEL contributions to the
mooring time-series, underway pCO2, and spatial survey data
used in these analyses were provided by NOAA’s Climate
Programme Office’s Global Carbon Cycle Programme (grants
GC05-288 and GC10-102), NOAA’s Ocean Acidification
Programme, and NOAA/PMEL. The Chá bă mooring off La
Push, Washington, was also supported by the Murdock Charitable
Trust, NANOOS (Northwest Association of Networked Ocean
Observing Systems, a Regional Association of NOAA’s US
Integrated Ocean Observing System Programme), UW/APL, UW
College of the Environment, and UW Provost’s Office. The
PRISM cruises were supported by the State of Washington
through UW Oceanography and APL. The NH10 mooring is
funded by NOAA through NANOOS, and by NSF through
CMOP (Science and Technology Center for Coastal Margin
Observation and Prediction OCE-0424602). Surface ocean pCO2
data collected on the NH10 mooring were supported by NSF
Chemical Oceanography OCE-0752576 awarded to BH and P.G.
Strutton. I. Kaplan, T. Klinger, and three anonymous reviewers provided valuable comments on early versions of the manuscript. This
is PMEL contribution number 4100.
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